Carrier status of annexin a5 m2 haplotype and obstetric risks

ABSTRACT

The present invention relates to determining the carrier status of Annexin A5 M2 haplotype of parents (both male and female) prior to and/or after pregnancy to minimize the risk of pregnancy complications, including, but not limited to, recurrent pregnancy loss (RPL), infertility, miscarriage, in vitro fertilization (IVF) failure, IUI failure, implantation failure, foetal growth restriction (FGR), small for gestational age (SGA) newborn, intra-uterine foetal death (IUFD), gestational hypertension (GH), pre-eclampsia (PE) and/or venous thromboembolism (VTE). Once M2 carrier status is determined, methods of intervention, including administration of low molecular weight heparin (LMWH) and/or other anti-coagulants can be administered either prior to and/or after pregnancy. Methods of detecting the carrier status as well as method of diagnosing and or predicting prognosis based on the M2 carrier status of a patient and/or couple is also contemplated.

FIELD OF THE INVENTION

The present invention relates to determining the carrier status ofAnnexin A5 M2 haplotype of parents (both male and female) prior to orafter pregnancy to minimize the risk of pregnancy complications,including, but not limited to, recurrent pregnancy loss (RPL),infertility (such as, for example, unexplained male infertility,unexplained female infertility, infertility of unknown origin) in vitrofertilization (IVF) failure, intrauterine insemination (IUI) failure,foetal growth restriction (FGR), small for gestational age (SGA)newborn, intra-uterine foetal death (IUFD), gestational hypertension(GH), pre-eclampsia (PE) and/or venous thromboembolism (VTE). Once M2carrier status is determined, methods of intervention, includingadministration of low molecular weight heparin (LMWH) and/or otheranti-coagulants can be administered either prior to and/or afterpregnancy. Further methods of increasing rates of live births and/or IVFimplantation and/or decreasing clinical miscarriage are alsocontemplated. Methods of detecting the carrier status as well as methodof diagnosing and or predicting prognosis based on the M2 carrier statusof a patient and/or couple is also contemplated.

BACKGROUND OF THE INVENTION

Thrombophilias are a major cause of adverse pregnancy outcome (Markoffet al, 2011) and there is increasing evidence to suggest that impairmentof placental vasculature increases the risk of recurrent pregnancy loss(RPL), intra-uterine foetal death (IUFD), gestational hypertension (GH),pre-eclampsia (PE), venous thromboembolism (VTE), foetal growthrestriction (FGR) and small-for gestational-age (SGA) newborns (Youniset al, 2003; Grandone et al, 2003; Chinni et al, 2009; Tiscia et al,2009; Grandone et al, 2010; Tiscia et al, 2012).

Normal pregnancy is an acquired hypercoagulable state and thereforewomen with a genetic predisposition to thrombophilia may developclinical signs of coagulation defects de novo during pregnancy or duringthe postpartum period (Rey et al, 2003; Chunilal et al, 2009). Thepredisposing role of hereditary thrombophilic factors has been reportedin several clinical studies (Rodger et al, 2010), and historically inthe majority of patients the hereditary factor has been Factor V Leiden(FVL) or Prothrombin (PTm) (Bick et al, 2000). However, in 2007 a newhereditary factor for RPL and additional thrombophilia-related obstetriccomplications was identified (Bogdanova et al, 2007; Chinni et al,2010). This defect, termed the M2 haplotype, is a sequence variation inthe core promoter of the annexin A5 ANXA5 gene. It consists of fourconsecutive nucleotide substitutions in the core promoter and results inreduced expression of ANXA5 in placentas from M2 haplotype carriers whencompared to non-carriers.

Annexin A5 is a member of the annexin protein family which share theproperties of binding calcium and phospholipids. It is distributedabundantly and ubiquitously, mostly in kidney, liver and placenta(Morgan et al, 1998). It is most abundant on the apical membranes ofplacental syncytiotrophoblasts, the interface between maternal andfoetal circulation. ANXA5 was originally named “placental anticoagulantprotein”. It has been extensively studied both in-vivo and in-vitro(Thiagarajan et al, 1990; Romisch et al, 1991). It has potentanticoagulant properties associated with the phospholipid-bindingactivity and is one of the few annexins to be found extracellularly(Gerke et al, 2005). The ability of ANXA5 to form two-dimensionalaggregates on cell membranes has led to the development of the ANXA5“protective shield” model that postulates that ANXA5 shieldsphospholipids at this site from availability for coagulation reactionsand thus contributes to the maintenance of blood fluidity in theplacenta.

Annexin 5 is deficient in placentas of patients with antiphospholipidsyndrome (APS), and antiphospholipid antibody-mediated reduction ofAnnexin 5 on vascular endothelium may also contribute to systemicthrombosis (Rand, 1999). Bogdanova et al (2012) revisited the annexin A5protective shield model and reported that preliminary genotypinganalysis of a cohort of 30 lupus anticoagulant patients (LAC-positive)with obstetric APS revealed that 11 out of 30 were M2 carriers andsuggesting a threefold relative risk to develop obstetricantiphospholipid antibodies (aPA).

In very preliminary data in examining placental tissue, Markoff et al(2010) suggested not only that the decreased ANXA5 expression inM2/ANXA5 placentas (including those from women with FGR and or PE) isthe result of carriage of the M2 haplotype, but that this may occurregardless of parental origin, with obvious consequences for embryonalinduced risk rather than wholly maternal. They observed that the normalANXA5 allele does not compensate for observed M2 allele-specificdecreased messenger RNA levels and suggested that unlike FVL and PTm,where paternal thrombophilic genes are not associated with RPL (Toth etal, 2008), the M2/ANXA5 acts via the embryo.

This led to a pilot study of 30 RPL couples where all other causes ofRPL had been excluded (including inherited thrombophilias and APS). Inthis small and not powered sampling, the study suggested that male andfemales in these RPL couples may have an equal and increased M2carriership when compared to control populations. The authors concludedthat paternal and maternal carriage of the M2/ANXA5 haplotype mayassociate with RPL and confer equal risks. They further hypothesizedthat M2/ANXA5 may be the first instance of a hereditary factor causingpregnancy pathology by affecting embryonic anticoagulation (Rogenhoferet al, 2012).

Ueki et al 2012 in their knockout murine model found significantreductions both in litter size and foetal weight in ANXA5-null mice(ANXA5-KO) and thus demonstrated that the maternal supply of ANXA5 tothe circulation was crucial for maintaining normal pregnancy. Theyfurther observed that cross-breeding of ANXA5-KO and WT mice showed onlylitters bred using ANXA5-KO females had reduced numbers of pups. Theyalso demonstrated that administration of heparin on pregnancy days 12,14 and 16 to ANXA5-KO mice significantly increased litter size.

However, when these animal studies were extended to humans, the use oflow molecular weight heparin showed no beneficial effect. For example,in Rodger et al. reported in the journal Lancet that “previouslypublished high-quality evidence” existed suggesting “no benefit ofantepartum low-molecule-weight heparin in women with previous pregnancyloss, women with previous non-severe or late-onset pre-eclampsia, orwomen with previous small-for-gestational-age birth between the 5th and10th percentile.” See, Rodger et al., “Antepartum Dalteparin Versus NoAntepartum Dalteparin For the Prevention Of Pregnancy Complications InPregnant Women With Thrombophilia (TIPPS): A Multinational Open-LabelRandomised Trial,” Lancet 2014 Nov. 8; 384(9955):1673-83. Therefore, theauthors designed an adequately powered study (that took 12 years toperform) to finally answer with statistical evidence this open question.Specifically, Rodgers et al. explained

-   -   Our randomised trial is the first to show that thrombophilic        women without previous venous thrombosis do not benefit from        antepartum low-molecular-weight-heparin. Our meta-analysis shows        that lower quality evidence suggests that low-molecular-weight        heparin might prevent recurrent severe placenta-mediated        pregnancy complications (severe or early-onset pre-eclampsia,        small-for-gestational-age birth <5th percentile, and placental        abruption) but we did not record this benefit in the subgroup        analyses of our trial.    -   Rodger et al., page 9.

The authors further stated:

-   -   This trial addresses a key therapeutic question in a large and        vulnerable patient group. The absence of benefit is an important        finding. The discovery of an association between thrombophilia        and pregnancy complications in the mid-1990s led to widespread        off-label use of low-molecular-weight heparin in pregnant        women-both with and without thrombophilia-who had previous        pregnancy complications. This off-label use has been fueled by        the emotional consequences of these complications combined with        expert opinion, consensus panels and small non-randomised        studies suggesting benefit. Antepartum low-molecular-weight        heparin is not a benign intervention; it can be complicated by        heparin-induced thrombocytopenia (albeit rarely), withholding of        epidural analgesia, and, as shown in our trial, increased minor        bleeding, allergic reactions, skin reactions, raised liver        transaminase concentrations, and the risk of induction of        labour. Additionally, up to 400 subcutaneous injections of the        drug per term of pregnancy is both a personal and financial        burden. Clinicians and patients can be reassured that dalteparin        use throughout the antepartum period does not lead to        significant changes in bone mineral density. Finally, the        continued belief in ineffective therapy hampers further research        for efficacious treatments for women at risk of venous        thromboembolism and pregnancy complications.    -   Rodger, page 8.

Thus, there is a need for improved methods of reducing pregnancycomplications associated with thrombophilia caused by ANXA5 M2haplotype.

SUMMARY OF THE INVENTION

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used as an aid in determining the scope of the claimed subject.

The present invention relates to a method of treating an M2 haplotypepregnancy, wherein a M2 haplotype pregnancy is identified when it isdetermined that either the biological mother or the biological father isa carrier of the ANXA5 M2 haplotype and then the mother of a M2haplotype pregnancy is administered an effective amount of ananticoagulant. Surprisingly and contrary to the results obtained inother clinical trials, if the anticoagulant is administered immediatelyprior to, at the same time, and/or immediately after conception,intrauterine insemination, embryo transfer and/or implantation, the rateof live births are substantially improved. In some embodiments, both thebiological mother and the biological father are found to be carriers ofthe A5 M2 haplotype.

Other embodiments of the invention include a method of reducingobstetric complications comprising identifying a M2 haplotype pregnancy,wherein said M2 haplotype pregnancy exists when either the biologicalmother or the biological father is a carrier of the ANXA5 M2 haplotypeand then administering to the mother of a M2 haplotype pregnancy aneffective amount of an anticoagulant, wherein said anticoagulant reducesthe risk of obstetric complications. Preferably, the anticoagulant isadministered immediately prior to, at the same time and/or immediatelyafter conception, intrauterine insemination, embryo transfer and/orimplantation. In other embodiments, the anticoagulant is administeredfor at least 4 weeks, for at least 8 weeks, for at least 12 weeks, orfor at least 16 weeks. Examples of obstetric complications include, butare not limited to: recurrent pregnancy loss (RPL), infertility, invitro fertilization (IVF) failure, intrauterine insemination (IUI)failure, foetal growth restriction (FGR), small for gestational age(SGA) newborn, intra-uterine foetal death (IUFD), gestationalhypertension (GH), pre-eclampsia (PE) and/or venous thromboembolism(VTE).

Other preferred embodiments of the invention include a method ofdetermining a M2 haplotype pregnancy, comprising identifying the M2haplotype carrier status either the biological mother or the biologicalfather, wherein said identification is determined based on genomicanalysis; recording the M2 haplotype carrier status of the biologicalmother and the biological father; reporting whether a M2 haplotypepregnancy exists if either the biological mother or the biologicalfather is a carrier of the ANXA5 M2 haplotype. Preferred methods ofdetection include sequencing, PCR and/or SNP detection techniques.

Once M2 carrier status is determined, methods of intervention, includingadministration of low molecular weight heparin (LMWH) and/or otheranti-coagulants can be administered either prior to and/or afterpregnancy. Preferably, the anticoagulant is administered immediatelyprior to, at the same time and/or immediately after conception,intrauterine insemination, embryo transfer and/or implantation. Thus,further methods of increasing rates of live births and/or IVFimplantation and/or clinical pregnancy and/or decreasing clinicalmiscarriage are contemplated. In other embodiments, the anticoagulantincreases the rate of implantation. In other embodiments, theanticoagulant decreases the rate of miscarriage prior to detection of afoetal heartbeat. For example, in preferred embodiments, the mother ofan ANXA5 M2 haplotype pregnancy is administered an anticoagulantimmediately after detecting pregnancy using methods to detect earlypregnancy as soon as it is established. In other embodiments, theanticoagulant is administered at the same time of embryo transfer in anIVF setting. In even further preferred embodiments, the anticoagulant isadministered prior to pregnancy or embryo transfer in an IVF setting. Inother embodiments, the anticoagulant is administered until the motherdelivers the baby.

In other preferred embodiments, the anticoagulant is low-molecularweight heparin (“LMWH”). In preferred embodiments, the anticoagulant isadministered as part of in vitro fertilization. The mother may beadministered LMWH at the time of embryo transfer, prior to embryotransfer or within days of embryo transfer during IVF treatment.Preferably, administration of an anticoagulant, such as LMWH, occurssimultaneously with embryo transfer. In other embodiments, the LMWH isadministered for at least 4 weeks, for at least 8 weeks, for at least 12weeks, or for at least 16 weeks. In other embodiments, the LMWH isadministered until the mother delivers the baby.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts an ANXA5 promoter structure as disclosed in Carcedo(2001), Biochem. J. 356, 571-579) and Bogdanova N, Horst J, Chlystun M,Croucher P J, Nebel A, Bohring A, Todorova A, Schreiber S, Gerke V,Krawczak M, Markoff A (SEQ ID NO: 1). A common haplotype of the annexin(ANXA5) gene promoter is associated with recurrent pregnancy loss. HumMol Genet 2007; 16: 573-78. As reported in Bogdanova, FIG. 1 shows thestructure of the ANXA5 gene core promoter region. The boundaries aremarked by vertical bars and are numbered according to the position ofthe first transcription start point (tsp1). Non-translated exon 1 isshaded in gray. Transcription factor consensus motifs are in smallprint, and abbreviations of the corresponding transcription factors aredisplayed in italics above the sequence information. NotI and BamHIrestriction sites are underlined and the sequence of the Z-DNA stretchin the promoter is given in italics. Nucleotides marking transcriptionstart points (tsp) are underlined. Regions important for promoterfunction (motifs A and B) cover nucleotide positions 295-311 and328-337. Nucleotides changed in the M2 ANXA5 promoter haplotype areprinted in bold and substituting nucleotides are given in bold capitalletters on top of the respective positions. In Bogdanova et al., theposition of M2 ANXA5 haplotype substitutions are designated as −19G→A,1A→C, 27T→C and 76G→A (see, for example, page 574, first paragraph under“Results”) and correspond to 243G→A, 262A→C, 288T→C and 337G→A as shownin FIG. 1 herein. Similarly, these same substitutions also have beenreferred to as: (1) G to A at a position which corresponds to nucleotide186 of SEQ ID No. 2 in US2012/0178156; (2) A to C at a position whichcorresponds to nucleotide 203 of SEQ ID No. 2 in US2012/0178156; (3) Tto C at a position which corresponds to nucleotide 229 of SEQ ID No. 2in US2012/0178156; and (4) G to A at a position which corresponds tonucleotide 276 of SEQ ID No. 2 in US2012/0178156.

DETAILED DESCRIPTION

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and drawings, and fromthe claims.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to a “polynucleotide”includes a mixture of two or more such polynucleotide molecules or aplurality of such polynucleotide molecules.

As used herein, the term “comprise” or variations thereof such as“comprises” or “comprising” are to be read to indicate the inclusion ofany recited integer (e.g. a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g. features,element, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein, the term “comprising” is inclusive anddoes not exclude additional, unrecited integers or method/process steps.

In embodiments of any of the compositions and methods provided herein,“comprising” may be replaced with “consisting essentially of” or“consisting of”. The phrase “consisting essentially of” is used hereinto require the specified integer(s) or steps as well as those which donot materially affect the character or function of the claimedinvention. As used herein, the term “consisting” is used to indicate thepresence of the recited integer (e.g. a feature, element,characteristic, property, method/process step or limitation) or group ofintegers (e.g. features, element, characteristics, properties,method/process steps or limitations) alone.

The invention will be described in more detail below.

A. Definitions

In the present invention, a “M2 haplotype pregnancy,” or an “ANXA5 M2haplotype pregnancy” is defined as a pregnancy where either thebiological mother and/or the biological father are carrier(s) for theannexin ANXA5 M2 haplotype. It is contemplated that a “M2 haplotypepregnancy” includes situations where the biological mother has yet tobecome pregnant. Moreover, in the situation of a surrogate mother, bothdonor egg and/or sperm should be screened as well as the recipientmother for her own risk.

In the present invention, “annexin A5 M2 haplotype,” “M2 haplotype,” or“ANXA5 M2 haplotype” (SEQ ID NO: 2) is defined as a substitution in theannexin (ANXA5) promoter, wherein the substitutions are:

-   -   (i) a point mutation G to A at a position which corresponds to        nucleotide 243 of FIG. 1;    -   (ii) a point mutation A to C at a position which corresponds to        nucleotide 262 of FIG. 1;    -   (iii) a point mutation T to C at a position which corresponds to        nucleotide 288 of FIG. 1; and    -   (iv) a point mutation G to A at a position which corresponds to        nucleotide 337 of FIG. 1.

In the present invention, “M1 haplotype,” “annexin A5 M1 haplotype,” or“ANXA5 M1 haplotype” (SEQ ID NO: 3) is also disclosed in WO 2006/053725and Bogdanova et al., and is characterized by the following twonucleotide exchanges (1) A to C at a position which corresponds tonucleotide 262 of FIG. 1, and (2) T to C at a position which correspondsto nucleotide 288 of FIG. 1.

In the present invention, an “anticoagulant” is defined as a drug usedto prevent dot formation or to prevent a clot that has formed fromenlarging. Anticoagulant drugs inhibit dot formation by blocking theaction of dotting factors or platelets. Anticoagulant drugs fall intothree groups: inhibitors of dotting factor synthesis, inhibitors ofthrombin and antiplatelet drugs. Anticoagulants may be administered atthe time (e.g., simultaneously with and/or during the same procedure) ofembryo transfer or IUI, for example, or shortly (for example, within 1day, within 2 days, within 3 days, within 4 days, within 5 days, within6 days, within 7 days, within 8 days, within 9 days, within 10 days,within 11 days, within 12 days, within 13 days, or within 14 days) afterconception, implantation, IUI, embryo transfer, becoming pregnant and/orof learning of the pregnancy. Anticoagulants may be administered for atleast 4 weeks, for at least 8 weeks, for at least 12 weeks, or for atleast 16 weeks. Anti coagulants may be administered until the motherdelivers the baby. Additionally, an anticoagulant may be administeredimmediately before (for example, within 1 day, within 2 days, within 3days, within 4 days, within 5 days, within 6 days, within 7 days, within8 days, within 9 days, within 10 days, within 11 days, within 12 days,within 13 days, or within 14 days) prior to becoming pregnant,conception, implantation, IUI and/or embryo transfer.

Preferred examples of anticoagulants include, but are not limited lowmolecular weight heparin (LMWH) or aspirin, and preferably low doseaspirin.

In specific preferred embodiments, Low Molecular Weight Heparin (LMWH)is administered to the pregnant mother. LMWH can be purchased by anumber of different commercial sources. In the present invention, LMWHis may be administered to a patient every day throughout pregnancy untilshortly before delivery based on the treating physician's judgment.Female carriers of the M2 haplotype should also receive LMWH followingdelivery and for 6 weeks thereafter to reduce the risk of venousthromboembolism (VTE). LMWH may be administered at the time (e.g.,simultaneously with and/or during the same procedure) of embryo transferor IUI, for example, or shortly (for example, within 1 day, within 2days, within 3 days, within 4 days, within 5 days, within 6 days, within7 days, within 8 days, within 9 days, within 10 days, within 11 days,within 12 days, within 13 days, or within 14 days) after conception,implantation, IUI, embryo transfer, becoming pregnant and/or learning ofpregnancy. LMWH may be administered for at least 4 weeks, for at least 8weeks, for at least 12 weeks, or for at least 16 weeks. LMWH may beadministered until the mother delivers the baby. Additionally, LMWH maybe administered immediately before (for example, within 1 day, within 2days, within 3 days, within 4 days, within 5 days, within 6 days, within7 days, within 8 days, within 9 days, within 10 days, within 11 days,within 12 days, within 13 days, or within 14 days) prior to becomingpregnant, conception, implantation, IUI and/or embryo transfer.

As used herein, the phrase “at the same time” is defined as within hoursof undergoing fertilization, IUI, conception and/or embryo transfer.Preferably, “within hours” is defined as within 30 mins, 1, 2, 3, 4, 5,6, 7, 8 9, or 10 hours of undergoing fertilization, IUI, conceptionand/or embryo transfer.

As used herein, “intrauterine insemination” or “IUI” refers to a type ofartificial insemination where a concentrated solution of sperm is placeddirectly in the uterus or vagina around the time of egg release from theovaries. IUI may be performed with or without hormones and/or otherdrugs (e.g., such as Clomid) to increase the number of released eggsfrom the ovaries.

In the present invention, “obstetric complications” are defined ascomplications arising during pregnancy due to thrombophilia and/orimpaired placental vasculature. Examples of obstetrics complications,include, but are not limited to: recurrent pregnancy loss (RPL),infertility, in vitro fertilization (IVF) failure, IUI failure, foetalgrowth restriction (FGR), small for gestational age (SGA) newborn,intra-uterine foetal death (IUFD), still birth, gestational hypertension(GH), pre-eclampsia (PE) and/or venous thromboembolism (VTE).

In the present invention, the anticoagulant is effective to increase therate of implantation. In other embodiments, the anticoagulant iseffective to decrease the rate of early miscarriage (i.e. prior to thedetection of a foetal heartbeat).

In the present invention, “infertility” is defined as the inability fora couple to become pregnant, even when attempting pregnancy bymonitoring ovulation and/or by being administered hormones and/or otherdrug (e.g., Clomid) to increase the chance of pregnancy. Examples ofinfertility include, and are not limited to unexplained maleinfertility, unexplained female infertility, and/or infertility ofunknown origin.

It will be understood that in the context of the embodiments describedherein, the female subject was made pregnant by the herein mentioned“biological father”. Thus, as used herein, the term “biological father”means the biological father of the human embryo of the herein definedfemale subject. In some embodiments, the female subject is alreadypregnant and is therefore the “biological mother” in other embodimentsthe female subject is not yet pregnant and is therefore the intended“biological mother.”

The term “intended biological father” therefore means that the femalesubject is not yet made pregnant by the human male subject, but that itis intended that the human female subject will be made pregnant by saidhuman male subject. During that time, also the female subject is the“intended” biological mother. Once the female subject was made pregnantby said human male subject, the “intended biological father” becomes the“biological father” and the “intended biological mother” becomes the“biological mother”.

The methods of the present invention therefore encompass situationswherein the female subject is not yet made pregnant by the intendedbiological father, i.e. the female and/or the intended biological motherand the intended biological father plan to test the predisposition ofthe female subject to obstetric complications prior to the pregnancy.This includes for example couples which plan to have a baby or femaleswhich plan to become pregnant, either by natural procreation or by invitro fertilization.

The methods of the present invention can be used to increase the ratesof live births, to increase the incidence of implantation, increase theincidence of clinical pregnancy, and/or decrease the rates of clinicalmiscarriage. In the present invention, the phrase “live births” isdefined as a successful delivery of a living baby regardless of whetherthe baby is full term.

“Clinical miscarriage” is defined as whether the fertilized embryo (forexample, during in vitro fertilization the embryo transfer) results in aclinical miscarriage or a live birth. Patients reach a “clinicalpregnancy” stage when pregnancy can be confirmed, such as by anultrasound scan detecting a foetal heart(s)

The present invention “implantation” refers to the ability (even iftemporary) of an embryo to adhere to the uterine wall.

The methods of the present invention further encompass situationswherein the female subject is already pregnant. In such cases, it mightstill be wanted to test the predisposition of the female subject toobstetric complications, either by way of testing a sample of thebiological father and/or by way of testing an embryonic sample of theembryo as such (for example by circulating foetal cells, by way ofchorion biopsy or by way of amniocentesis, both resulting in samples ofembryonic origin). Additionally, but not exclusively, it is alsoenvisaged to test the (intended) biological mother.

Provided that the fertilization is conducted in vitro, i.e. by way of anin vitro fertilization, it is also envisaged to analyse a single cellsample obtained before or during the morula stage of the in vitrofertilized embryo, prior to its implantation into said female subject.

The “morula stage” denotes the 16 cell stage of human embryogenesis.“Before or during the morula stage” means that it is also envisaged toobtain one single cell prior to the 16-cell stadium, for example duringthe 6-8 cell stadium of human embryogenesis.

In vitro fertilisation (IVF) is a well-known process by which egg cellsare fertilised by sperm outside the womb, in vitro.

It is also envisaged that females which intend to become pregnant by asperm donor, make use of the methods of the present invention in orderto test their predisposition to obstetric complications by way oftesting the respective sperm donor sample before it is used for the invitro fertilization of the respective female subject and then beingtreated with an anticoagulant prior to, at the same time and/orimmediately after conception, intrauterine insemination, embryo transferand/or implantation. The present invention and in particular the methodsof the present invention therefore also encompass a stratificationmethod for selecting a sperm donor, which is not a carrier of the riskhaplotype M2.

Thus, by way of the methods of the present invention it is possible todetect and subsequently select a sperm donor who, in all likelihood,will not contribute to the predisposition of the respective femalesubject to obstetric complications. These stratification methods makeparticularly sense when a maternal sample of the mother was alreadytested to be no carrier of the risk haplotype M2, because in such casesit is of importance to test whether the intended biological father (forexample the sperm donor) is a carrier of the mentioned risk haplotypes.If so, then it might be reasonable to select a different sperm donor,preferably sperm donor who is also no carrier of the risk haplotypes M2.

In the same way, it is reasonable and therefore particularly envisagedin the embodiments of the present invention to test the intendedbiological father or the biological father in situation where thebiological mother (or the intended biological mother) is no carrier ofthe risk haplotype M2 (as tested in a maternal sample).

It is also envisaged that females which intend to become pregnant by adonor eggs, make use of the methods of the present invention in order totest their predisposition to obstetric complications by way of testingthe respective egg donor genotype before it is used for the in vitrofertilization. The present invention and in particular the methods ofthe present invention therefore also encompass a stratification methodfor selecting an egg donor, which is not a carrier of the risk haplotypeM2.

Thus, by way of the methods of the present invention it is possible todetect and subsequently select egg donors who, in all likelihood, willnot contribute to the predisposition of the respective female subject toobstetric complications. These stratification methods make particularlysense when a maternal sample of the mother was already tested to be nota carrier of the risk haplotype M2, because in such cases it is ofimportance to test whether the intended biological egg donor is acarrier of the mentioned risk haplotypes. If so, then it might bereasonable to select a different egg donor, preferably an egg donor whois also no carrier of the risk haplotypes M2.

In the same way, it is reasonable and therefore particularly envisagedin the embodiments of the present invention to test the intendedbiological egg donor and/or the biological father in situation where themother (or the intended mother) is no carrier of the risk haplotype M2(as tested in a maternal sample).

In the present invention, both the mother but also the father cancontribute to a predisposition of the female subject to obstetriccomplications. Accordingly, even if the female subject as such is not acarrier of the risk haplotype (tested in a maternal sample), thebiological father and/or the intended biological father can stillcontribute to the above mentioned predisposition and should, therefore,be tested as well.

Provided that either the biological father of the embryo or thebiological mother of the embryo is a heterozygous carrier of the riskhaplotype, it is also possible to test a sample of said embryo (e.g. achorion biopsy sample or a single cell sample described herein) in orderto test whether it is carrier of the risk haplotype or not (for examplein case of an in vitro fertilization). Provided that either thebiological mother or the biological father is a homozygous carrier ofthe risk haplotype, it appears unnecessary to test the embryo as well,as the heterozygous presence of the risk haplotypes M2 is alreadyindicative for a predisposition of the respective female subject toobstetric complications. Provided that the biological father isuntraceable or unknown, and further that the female subject (in thatcase the biological mother) is not a carrier of the M2 risk haplotype,one might still want to test the predisposition of the respective femalesubject to obstetric complications. In that case it is envisaged to testa sample which originates from the embryo (e.g. circulating foetal bloodcells, chorion biopsy and/or amniocentesis sample). It will beunderstood, however, that an embryonic sample should preferably not beobtained solely because the female subject intends to test itspredisposition to obstetric complications. It is rather envisaged totest in samples which originate from the embryo only then, when suchsamples are already at hand for other reasons.

In the present invention, recurrent pregnancy loss (RPL) is typicallycharacterized as the occurrence of two or more pregnancies that end inmiscarriage of the foetus. Said two or more pregnancies occur eitherconsecutively or intermittently, consecutively being preferred.

In the present invention, pre-eclampsia (PE) is a medical condition inwhich hypertension arises in pregnancy (pregnancy-induced hypertension).

Foetal growth restriction (or foetal growth retardation) is a conditionin which a foetus does not grow appropriately. FGR should be suspectedfor example when the fundal height is more than 3 cm less thanpredicted.

It is envisaged that in the methods of the present invention, saidsample obtained from the individual (whether mother or father) from ablood sample, a sperm sample, a tissue sample, or a cell sample. It willbe understood that any biological sample will be suitable as long as therespective sample contains genetic material which allows thedetection/diagnosis which are subject of the methods of the presentinvention.

Such a sample may be obtained via biopsy such as needle biopsy, surgicalbiopsy, via any kind of smear technique, for example by use of a buccalswab, etc. or others. The skilled person is well aware of further meansand methods enabling him or her to obtain a sample containing geneticmaterial from a human subject.

The skilled person is well-aware how to avoid or circumvent suchcontaminations “the corresponding standards are for example summarizedin the “General standards and guidelines for prenatal testing areavailable from the American College of Medical Genetics (2006 Edition ofStandards and guidelines for clinical genetics laboratories,http://www.acmg.net/Pages/ACMG_Activities/stds-2002/g.htm”.

B. Methods of Detecting

As already disclosed in WO 2006/053725 and Bogdanova N, Horst J,Chlystun M, Croucher P J, Nebel A, Bohring A, Todorova A, Schreiber S,Gerke V, Krawczak M, Markoff A. A common haplotype of the annexin(ANXA5) gene promoter is associated with recurrent pregnancy loss. InHum Mol Genet 2007; 16: 573-78, the risk haplotype M2 which can bedetected in the human ANXA5 promoter, is characterized by the followingfour nucleotide exchanges:

-   -   (1) G to A at a position which corresponds to nucleotide 243 of        FIG. 1;    -   (2) A to C at a position which corresponds to nucleotide 262 of        FIG. 1;    -   (3) T to C at a position which corresponds to nucleotide 288 of        FIG. 1; and    -   (4) G to A at a position which corresponds to nucleotide 337 of        FIG. 1 (SEQ ID NO: 2).

The risk haplotype M1 which can also be detected in the human ANXA5promoter, is also disclosed in WO 2006/053725 and Bogdanova et al., andis characterized by the following two nucleotide exchanges (1) A to C ata position which corresponds to nucleotide 262 of FIG. 1, and (2) T to Cat a position which corresponds to nucleotide 288 of FIG. 1 (SEQ ID NO:3). FIG. 1 depicts an ANXA5 promoter structure as disclosed in Carcedo(2001), Biochem. J. 356, 571-579) and Bogdanova et al (SEQ ID NO: 1).

Means and methods to determine and/or to detect the M2 haplotypes arewell-known (see for example WO 2006/053725 and Bogdanova et al.) andadditionally disclosed in detail herein.

“Nucleic acid detection techniques” are well-known to the skilled personand include inter alia any kind of PCR-based techniques or any othersuitable technique which allows the identification of the nucleotideexchanges which characterize the risk haplotype M2. Such methods aredescribed herein (see the examples) and are also published for examplein WO 2006/053725.

Said techniques may be selected from the non-limiting group consistingof hybridization techniques, nucleic acid sequencing, PCR, restrictionfragment determination, single nucleotide polymorphism(SNPs)-determination, LCR (ligation chain reaction) or restrictionfragment length polymorphism (RFLP)-determination, to name some.

Corresponding examples and further details may be obtained from standardtechnical advice literature (like Sambrook, Russell “Molecular Cloning,A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001);Ausubel, “Current Protocols in Molecular Biology”, Green PublishingAssociates and Wiley Interscience, N.Y. (1989), or Higgins and Hames(Eds.)). As documented in the examples of WO 2006/053725, a furthersuitable method is the restriction fragment determination or the RFLPmethod, comprising the determination of a BamHI restriction site. Asshown in WO 2006/053725, the absence (BamHI′) or the presence (BamHI+)of a BamHI restriction site is determined, and is indicative for theabsence or presence of a point mutation as defined herein. Details onthis method are given in the appended examples of WO 2006/053725.

In one embodiment, a relevant DNA-stretch may be amplified from genomicDNA by PCR-technology. Potential primers to be employed comprise, butare not limited to, the primers as provided in SEQ ID NO: 4 (ANX5.P.F;SEQ ID NO: 22 of WO 2006/053725) and SEQ ID NO: 5 (ANX5.exl. R; SEQ IDNO: 23 of WO 2006/053725). The person skilled in the art is readily inthe position to deduce further primer pairs or primers to be employed inorder to amplify relevant stretches of the herein defined annexin A5(ANXA5) promoter or of its fragments. After the amplicon is obtained(see also experimental part) it can be digested (restriction digest)with the restriction enzyme BamHI (which can be obtained from varioussuppliers, inter alia: Roche Applied Science, Mannheim, Germany; MBIFermentas, St. Leon-Rot, Germany; New England Biolabs, Frankfurt amMain, Germany. Again, details are given in the experimental part. Afterthis digest, to be carried in accordance with methods well-known in theart (see inter alia Sambrook/Russel, 2001, (log.cit.)), further analysisof the BamHI/BamHI+restriction site can be carried out by knowntechniques, like gel analysis, e.g. agarose gel analysis.

A further technique which is particularly envisaged in the context ofthe present invention is the SNP detection technique established by IHGPharmaco. Said technique is sufficiently explained in WO 2006/038037 andin U.S. Pat. No. 7,803,545 and is hereby incorporated by reference inits entirety.

Specifically, in genotyping using IHG technology, a sample of nucleicacid is obtained from the subject, and the gene segment containing thepolymorphic site is amplified to provide a population of ampliconsbearing the sequence of the gene segment. Typically, this amplificationof the gene segment is accomplished by PCR using a pair of primers whichflank the said gene segment. Suitable primers are selected which arespecific for the gene segment under consideration. Primers are selectedto amplify a gene segment which is of the order of from 90 to 400 basesin length, and preferably of the order of 100 to 150 base pairs inlength. It is normally preferred that the polymorphic site is located inthe central region of the gene segment, that is to say approximately inthe central third of the gene segment. PCR amplification of the genesegment will result in a population of double stranded amplicons, as iswell known. More details of the procedure which may be used for PCRamplification can be found in WO 93/19201.

Where the nucleic acid under examination is mammalian genomic DNA, asample of the DNA is obtained from an individual or other object whosegenotype for a specific characteristic it is wished to study. (The term“individual” is intended to include a foetus.) DNA can be extracted fromall nucleated cells. Typically, the DNA is obtained from peripheralblood cells for convenience. Foetal DNA can be obtained from placentalcells or amniotic fluid. Other sources of DNA include hair follicles,mummified bodies, etc. The DNA may be isolated by any appropriatemethod, for example by the rapid salting out method described by Milleret al (Miller, S., Dykes, D. and Polesky, H. (1988) “A simple saltingout procedure for extracting DNA from human nucleated cells”; Nucl.Acids Res. 16:1215). Alternatively, the DNA may be isolated as cDNA frommRNA by reverse transcription.

A population of an IHG molecule which has a sequence corresponding tothe gene segment, but modified as discussed herein to include controllednucleotide substitution, deletion, insertion or combination thereof isalso provided. Typically, the IHG population is prepared byamplification using, for example PCR. Again, more details of theprocedure which may be used for PCR amplification can be found in WO93/19201, which is hereby incorporated by reference in its entirety. Theprimers chosen for the PCR are selected to provide amplification of theIHG molecule. PCR amplification will result in a population of doublestranded IHG amplicons. The IHG may preferably be substantiallyidentical in length to the gene segment under consideration(disregarding any necessary inserted or deleted bases of the IHG), ormay be a different length, for example shorter than or longer than thegene segment. However, if the gene segment and IHG are of differentlength (disregarding any necessary inserted or deleted bases of theIHG), there must be a sufficient degree of overlap to permitheteroduplex formation between the amplified populations of the genesegment and the IHG. The primers used in amplifying the IHG and the genesegment respectively may be the same or different. Typically, however,the same primers are used, resulting in amplified IHG and gene segmentwhich are of substantially the same length (disregarding any necessaryinserted or deleted bases in the IHG). As taught in WO 93/19201, theprimers may be labelled.

PCR amplification of the gene segment and the IHG may be accomplished inthe same or separate vessels (“mixed” or “separate” PCR respectively).It is preferred to conduct amplification separately and then to combineor pool the amplified populations of the gene segment and the IHG inorder to permit heteroduplex formation to proceed.

Heteroduplex formation between the combined populations of IHG and thegene segment which contains the polymorphic site is accomplished byfirst heating the combined population of IHG and gene segment in orderto separate the double stranded DNA into single stranded DNA and thencooling to permit heteroduplex formation, as described for example inWO-A-93/19201.

The heteroduplexes formed are separated according to their molecularconformation which affects their apparent, but not actual, molecularweight. This may be achieved by, for example, electrophoresis. Theseparation is typically effected on a gel which does not fully denaturethe nucleic acid, such as a non-denaturing polyacrylamide gel.Electrophoresis is conducted under conditions which effect a desireddegree of resolution of the duplexes. A degree of resolution thatseparates duplexes that differ in “apparent size”-resulting from theirdifferent molecular conformations—by as little as about 10 bp is usuallysufficient. Size markers may also be run on the gel to permit estimationof the mobility and thus the apparent size of duplexes. In addition, oralternatively, a control DNA molecule having a sequence whichcorresponds to the known allele of the gene under consideration can alsobe separately amplified using PCR and allowed to form heteroduplexeswith the IHG being used, with the resultant sample then being run on thegel to provide markers on the gel for the different heteroduplexes whichresult.

The distribution, i.e. the resolution pattern, of the heteroduplexeswill be allele-specific. This resolution pattern or PCR fingerprint cannext be visualised. Where the PCR primers have been labelled, this labelmay be revealed. A substrate carrying the separated labelled duplexes iscontacted with a reagent which detects the presence of the label. Wherethe PCR primers were not labelled, the substrate bearing the PCRfingerprint may be contacted with, for example, ethidium bromide orSYBR™ green (available from Molecular Probes) and the nucleic acidfragments visualised under ultraviolet light; alternatively, theheteroduplexes may be visualised with silver staining.

Additional methods of detecting M2 haplotype status, include, but arenot limited to methods utilizing a fluorescent molecule and/or a solidphase. Preferred solid phase structures can be beads and/or plates.Solid phases can comprise plastic, silicon, glass, polystyrene,aluminium, steel, iron, copper, nickel, silver, gold, nitrocellulose ornylon. Additionally, oligos at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, at least 27, at least 28, at least29 and/or at least 30 nucleotides in length can be used to detect theANXA5 M2 haplotype. Moreover, methods of detecting ANXA5 M2 haplotypestatus can include methods utilizing Next Generation Sequencingtechniques. These methods of detecting are all within the skill in theart.

EXAMPLES Example 1. Study Population

Study patients were recruited between March 2012 and February 2013 frompatients attending five fertility clinics. Informed consent was obtainedfrom all patients. During this period 314 patients (157 couples)presented with at least 1 previously failed IVF cycle (average 1.9 IVFand 0.2 IUI). A detailed clinical history was obtained and thegenotyping for presence or absence of carriage of the M2/ANXA5haplotype, formed part of the diagnostic investigations for infertility.The mean age of females was 36.3 years (Range 23 to 49) and that oftheir male partners 38.6 years (Range 23 to 64). Average Body Mass Indexof the females was 25.5 (range, 19 to 40.5) and that of their malepartners was 33.7 (Range 21 to 36). The selection of patients forscreening was based on their prior history and the patient's willingnessto be tested, following the detailed nature of the study being providedto them at consultation. Female patients were screened forantiphospholipid antibodies. With regard to their infertility status,the majority of the male population had oligospermia (48%),astheno/oligoasthenospermia (27%), or azoospermia (13%). These variedaccording to carrier status with an incidence in the non-carriers of41%, 26% and 11%, respectively; and for the carriers 35%, 12%, and 12%,respectively. With regard to the female population, the most prevalentwas unexplained (27%), poor ovarian reserve (17%), PCOS (11%), andendometriosis (6%). The variation according to carrier status with anincidence in the non-carriers was 30%, 16%, 16% and 3%, respectively;and for the carriers 26%, 9%, 18%, and 8%, respectively.

The majority of these patients were White British (77% males and 75%females), Indian/Pakistani (8%) the remainder being of diverseethnicities. As a whole this cohort is representative of the demographyof the UK and Eire.

Example 2. Collection of DNA and Determination of M2 Carrier Status

Deoxyribonucleic acid (DNA) was collected from couples either by a bloodsample (the first cohort), with the remaining cohort undergoing buccalcell analysis on specific collection paper. Extensive laboratory testswere undertaken to ensure the transfer to buccal cell collection causedno deterioration in the quality of the data. DNA was extracted fromwhite blood cells using QIAmp DNA blood mini kit (Qiagen GmbH, Hilden,Germany) or from elution off the collecting paper. PCR reactions werecarried out on 100 ng of genomic DNA isolated from blood samples usingthe Qiamp blood mini kit or from purified collecting paper punchesincluded in subsequent PCR reactions as template. PCR reactions werecarried out using Biotaq Polymerase (Bioline. Bioline Reagents Limited,London, Great Britain) in a volume of 25 μl containing 10×NH₄ ReactionBuffer: 160 mM (NH₄)₂SO₄, 670 mM Tris-HCl (pH 8.8), 50 mM MgCl₂ (finalconcentration 1.5 mM), 50 pM primer (forward and reverse), 200 M dNTP's,polymate additive (Bioline) and 2.5 units Biotaq polymerase. PCR cyclingconditions were as follows, 94° C. for 45 seconds, 30 cycles of 94° C.for 30 seconds-60° C. for 30 seconds-68° C. for 1 minute and a finalextension step of 7 minutes. PCR products were purified using standardcolumn purification methods using a Zymo ZR-96DNA Clean and Concentratorkit (Zymo Research Corporation. Irvine, Calif., USA). Purified ampliconswere sequenced using ABI BigDye terminator chemistry v3.1 using standardconditions and electrophoresed on an ABI 3730×1 DNA analyser. Traceswere analysed and genotyped using ABI Seqscape v 2.5. (AppliedBiosystems, Foster City, Calif., USA). The presence of the M2 haplotype(a set of four consecutive nucleotide substitutions in the ANXA5 genepromoter: 243G/A [rs112782763], 262A/C [rs28717001], 288T/C [rs28651243]and 337G/A [rs113588187]) was investigated (SEQ ID NO: 2). When only twoof the four variants (262A/C, 288T/C) were present, the haplotype wasdefined as M1 (SEQ ID NO: 3).

All genotype calls were made using the Seqscape software (AppliedBiosystems, Foster City, Calif., USA) with a 25% mixed base callingthreshold. Seqscape was programmed to analyse nucleotide variations at 4specific bases as described in the literature. Results were generated inthe form of a mutations report detailing mutations across the region ofinterest. Report production was carried out by means of an in house LIMSsystem (Laboratory Information Management System), which was programmedto only allow certain combinations of mutation. Any sample which gave anunexpected result was flagged by the LIMS system and checked by anoperator before repeating the test on a fresh sample.

Example 3. Genotyping and Statistical Analysis

Patients who were heterozygous or homozygous carriers of the M2/ANXA5haplotype were recorded as affected hetero or homozygous carriers. Testsfor deviations from Hardy-Weinberg were performed using the method ofGuo and Thompson, 1992 (also used by Bogdanova et al 2007 and Rogenhoferet al 2012). We performed this test within the male and female groupsconsidered separately and overall. We also tested excluding theindividuals of non-UK ethnicity to see whether this affected theresults.

To check whether the significant deviation from Hardy-Weinbergequilibrium (HWE) observed in the female subgroup could be attributed tochance we subsampled 155 individuals at random from the entire set(males and females combined) and estimated the p-value for deviationfrom HWE using the same method and recorded the p-value. We performedthis procedure 1000 times, and of these only 3 p-values recorded weremore extreme than those observed for the all-female group, thussuggesting that the deviation from HWE in females is real and notattributable to chance.

The controls used for comparison are those used by Rogenhofer et al 2012from a population control sample drafted from the PopGen biobank atUniversity Clinic Schleswig-Holstein Kiel (n=533). PopGen populationcontrols were from Northwest Germany and were healthy subjectsidentified through official population registers (Krawczak et al 2006).The sample used in this study comprised approximately equal numbers ofmen and women distributed among three age groups (18-30, 30-50, and50-80 years). The cohort of Muenster fertile controls were anonymisedindividuals from the Institute's registry (Rogenhofer, et al 2012), allwith successful pregnancies and no documented history of recurrentpregnancy loss.

Example 4. Patient Genotype Frequencies

Of the 314 patients (6 patients were not genotyped because 4 males (2azospermia, 1 oligospermia 1 aged 65); 2 females: 1 early menopause 1menopause) and 157 couples the overall patient carriage rate was 25%(N=78) and of similar incidence in females (24% N=37) and males (27%N=41). However, of couples there was a high (44%; n=69) incidence of M2carriage (defined as one or both partners being M2 carriers orhomozygotes). None of these patients tested positive forantiphospholipid syndrome (APS). Amongst these carrier couples weresmall subsets of couples in which one partner was a non-carrier and onewas homozygous (4% N=7), or both partners were carriers (4% N=6), or onepartner was a carrier and one homozygous (2% N=3). There were 9 femalehomozygotes and one male. The genotype distribution of male and femalepatients is shown in Table 1.

TABLE 1 Genotype Distributions of Male/Female Partners Genotype Male %Female % N 153 155 N/N 88 58% 97 63% N/M1 24 16% 21 14% M1/M1 0  0% 0 0% N/M2 37 24% 27 17% M1/M2 3  2% 1  1% M2/M2 1  1% 9  6% Totalgenotypes 306 100%  310 100%  The genotypes N M1 M2 refer to haplotypesin the ANZXA5 gene promoter: N = normal/Wild type; M1 comprises 262A→Cand 288T→C (six heterozygotes); M2 comprises 243G→A, 262A→C, 288T→C and337G→A (16 heterozygotes).

The genotypes expected under HWE and observed genotype frequencies arepresented in Table 2 for males and females respectively. There is nosignificant deviation from HWE in males, but there is significantdeviation amongst females (p=0.005). Restricting the analyses toindividuals of British or Irish ethnicity gave similar results (data notshown).

TABLE 2 Observed and HWE Expected Genotype Counts for Males and Females,with estimated p-values for the test of departure from HWE computed viaMarkov Chain Monte Carlo (MCMC). Males Males Females Females ObservedExpected Observed Expected N|N 88 (57.5) 91.8 97 (62.6) 94.5 N|M1 24(15.7) 20.9 21 (13.5) 17.2 M1|M1 0 (0)  1.2 0 (0)  0.8 N|M2, M1|M2 40(26.1) 36.2 28 (18.1) 39.2 M2|M2 1 (0.7) 2.9 9 (5.8) 3.4 Total 153(100)   153 155 (100)   155 Estimated P-value NS 0.00517 P-value StdError 0.0001 0.0000 NS = not statistically significant. Note Values arenumber (percentage)

The genotype frequencies of ANXA5 gene promoter haplotypes in IVFcouples included in the current study and two different control groupsare shown in Table 3. The abundance of the M2 haplotype was enriched inboth male and female IVF patients compared to both the Muenster controls(female) and the PopGen controls (male and female).

TABLE 3 Genotype Frequencies of ANXA5 Gene Promoter Haplotypes in UK IVFCouples & Two Different Control Groups Muenster IVF Male and Femalepatients Fertile Controls^(a) PopGen^(b) Males Males Females FemalesFemale Male & Female Controls Observed Expected Observed ExpectedObserved Expected Observed Expected N | N 88 (57.5) 91.8 97 (62.6) 94.5356 (71.2) 343.6 415 (77.9) 413.3 N | M1 24 (15.7) 20.9 21 (13.5) 17.2 87 (17.4) 99.5 35 (6.6) 47.8 M1 | M1 0 (0)   1.2 0 (0)   0.8 16 (3.2)7.2  1 (0.2) 1.5 N | M2, 40 (26.1) 36.2 28 (18.1) 39.2 31 (6.2) 48.4  77(14.4) 69.0 M1 | M2 M2 | M2 1 (0.7) 2.9 9 (5.8) 3.4 10 (2.0) 1.4  5(0.9) 1.4 Total 153 (100) 153 155 (100) 155 500 500 533 533 Note Valuesare number (percentage) Expected values correspond to those expectedunder Hardy-Weinberg equilibrium

The IVF female patients were not in HWE (p=0.0052) owing to the excessof M2 heterozygotes but particularly M2 homozygotes (9 observed v.3.4expected). To check whether the significant deviation from HWE observedin the female subgroup could be attributed to chance we subsampled 155individuals at random from the entire set (males and females combined)and estimated the p-value for deviation from HWE using the same methodand recorded the p-value. We performed this procedure 1000 times, and ofthese only three p-values recorded were more extreme than those observedfor the all-female group, thus suggesting that the deviation from HWE infemales is real and not attributable to chance

The patients' previous IVF, IUI and pregnancy histories are shown inTable 4. The numbers of previous failed IVF cycles were highest incouples who had one partner a homozygote and one non-carrier (mean 3.1previous IVF) and in couples where the male partner was a carrier (mean2.1 previous IVF).

TABLE 4 Patients' Previous IVF and IUI Cycles and Pregnancy HistoriesTotal Live Time of last miscarriage Classification Couples IVF cyclesIUI Pregnancies miscarriages births (gestational weeks)^(a) Couples 69(2.0) 23 (0.9) (0.7) 4 (10.1, 5-23) (1 or both 191 63 50 (n = 17)partners a M2 carrier or homozygote) Male only carrier 31 (2.1) 3 (1.1)(0.8) 1 (9.6, 7-22) 66 33 26 (n = 11) Female only 22 (1.6) 12 (0.8)(0.7) 2 23 carrier 36 (all same 17 15 patient) Homozygous 7 (3.1) 3(1.3) 6 0 5, 9 partner (6 female 22 9 (1 female had 4, 1 male) and non-(1 female had 4) 1 female had 2) carrier partner Both partners 6 (1.9) 5(0.6) 3 1 6, 7, 15 carriers 17 5 1 partner 3 (1.7) 0 3 3 0 7, 15, veryearly homozygote, 5 1 partner carrier Non-carrier 88 (1.9) (0.2) (0.9)(0.6) 13 9 weeks, 5-26 couples 153 12 83 53 n = 25 * Pregnancy Loss Notevalues are numbers, (mean) or (mean, range).

Previous live births were very low in all carrier/homozygous groups(range 0-4) and a slightly higher incidence was observed in non-carriercouples (N=13). The patients' most recently reported miscarriage incarrier couples occurred at a mean of 10.1 weeks (range 5 to 23 weeks)in the 17 miscarriages where date of loss was reported. In non-carriercouples miscarriage (N=53) occurred at a mean of 9 weeks (range 5-26) inthe 25 of 53 miscarriages.

Example 5. Male Infertility and M2 Carriage Frequency

Overall 63 of 157 males (40%) had associated infertility factors.Carriage incidence in this group was 27% (N=17). Overall, oligospermiawas the most frequent finding (40% N=25 of the infertile males) followedby oligoasthenoteratozoospermia (OATS-13% N=8 of the infertile males).

Of 157 female patients, 93 (59%) had a diagnosis of infertility otherthan unexplained or male factor. Additionally, 25 of the 93 (27%) with adiagnosis were also found to be M2 carriers. Unexplained, poor ovarianreserve/ovulation failure often linked to age, plus PCOS are the mostfrequently cited causes of infertility in both groups. However, maleinfertility is cited as the primary cause of infertility in the couplein 21% of the non-carrier group but noted in only 1 of the group of 37patients who carried the M2 haplotype. Six out of 17 PCOS cases (35%)were also carriers.

Example 6. Unexplained Infertility and M2 Carriage Frequency

104 patients (33%) presented as having no explanation for infertility.Of these, 38 patients (37%) were identified as M2 carriers; 25 male(24%) and 13 female (13%). There were 9 female homozygotes (6% of allfemales). There was also one male homozygote aged 49 for whom the couplehad no other known diagnosis although his female partner had had 2 IVFcycles which had resulted in miscarriage.

Example 7. Preliminary Results

Carriership of the haplotype M2/ANXA5 in this cohort of patient coupleswas 44%, representing a very high incidence. Furthermore it was presentin 27% of male infertility patients, 27% of female infertility patientsand in 37% of patients with previously unexplained reasons forinfertility. Of the patients who carried the M2 haplotype in the presentstudy, none tested positive for APS. Genotype M1/MI was absent in theRPL cohort and rare in controls. Genotype M1/M2 was not observed in therecurrent pregnancy loss cohort and seen only in a total of 8 fromcontrol groups and in only 4 patients in this IVF cohort. However theincidence of homozygote M2 female patients was elevated at 6% in thiscohort and one male M2 homozygote was recorded.

Female homozygote frequency was three times higher than that reportedfrom other control groups and double that of recurrent pregnancy lossfemales (Rogenhofer et al 2012).

We justify the use of the PopGen and Muenster controls as Nelis et al(2009) concluded that four areas could be identified namely 1) Centraland Western Europe, 2) the Baltic countries, Poland and Western Russia,3) Finland, and 4) Italy, which if not corrected for the interpopulationdifferences would affect the significance of disease gene associations.The incidence in controls from published studies from Germany, SouthernItaly and Bulgaria—representative of three of these regions—have allshown consistency in the M2 haplotype frequency. The majority of the IVFpatients were White British (77% males 75% females), which correspond tothe Central and Western Europe region. We had no Finnish patients andanalysis with and without the subset of Indian/Pakistani and othersstill showed the significant departure from HWE in females but not inmales mainly due to the abundance of M2 homozygotes.

In terms of ethnicity we found M2 carriers in a wide range ofethnicities including Jewish, Turkish and Middle East patients inaddition to Indian and Pakistani patients. The possible differences incarriage rate and clinical effects in these ethnicities warrants furtherinvestigation since there may be significant differences in incidenceand pathology. The incidence in the Caucasian populations of Europe iswell established (Markoff et al, 2011) and Myamura et al (2011) reportedthat carriage of the haplotype resulted in similar risks for recurrentpregnancy loss in the Japanese population as that observed in thepopulations of central Europe; but the population incidence is lower(5.5 versus 15%). Thus further study of different ethnicities other thanwhite Europeans and Japanese is warranted.

Any impairment of embryonic coagulation is of particular importance inIVF practice since the focus is often on managing and providing forhealthy gametes and embryos, selecting for optimal embryo viability andensuring a healthy uterus able to sustain a pregnancy. However, althoughthe largest single cause of miscarriage is believed to be the aneuploidembryo, other factors are clearly of significance, especially in RPLcases, where it can remain an issue even after the transfer of euploidembryos following IVF. The relatively recently discovered genetic factorM2/ANXA5 is alone in influencing placental function via adverse effectson embryonic anticoagulation and if undetected could negate theconsiderable work and cost incurred to establish a healthy pregnancy viaIVF. In our study there were a significant number of patients equallydistributed between male and female where M2 carriage was either anadditional factor to those already determined, or it was present in asignificant number of patients with no other infertility diagnosis.There is a growing body of evidence of the risks of carriage of theM2/ANXA5 haplotype to maternal health (RPL, VTE, PE, GH APS: Tiscia etal, 2009; Grandone et al, 2010; Bogdanova et al, 2012). Bogdanova et al(2012) postulate that carriage of the M2/ANXA 5 haplotype leads to areduced ANXA5 cover of exposed phosphatidylserine surfaces, and thisreduced shielding would allow coagulation factors to compete forphospholipid binding. Secondly, there would be greater exposure ofphospholipid antigenic factors that would then lead to aPA developmentwhich in turn would further disrupt the ANXA5 shield. Sifakis et al(2010) demonstrated significant differences in mRNA expression betweennormal and FGR pregnancies but no differences in ANXA5 protein levels.However, the authors did not genotype their samples for M2/ANXA5.

Additionally, the identification of a subset of patients before IVFtreatment that are ANXA5 carriers is important since from this studytheir IVF cycle failure rate is higher than for non-carriers. We reporthere a single male homozygous patient with no other infertilitydiagnosis whose female partner had had 2 previous failed IVF cycles.Thus identifying and treating female patients who are themselves M2carriers or whose male partner is a carrier may assist in reducing theincidence of small for gestational age (SGA) by mitigating the adverseeffects on embryonic anticoagulation.

Since the defect is conveyed embryonally and affects embryonicanticoagulation and the risk is independent of any specific parentaltransmission, that is, it can be embryonally induced if the transmissionis either maternal or paternal (or both), screening of both partnerspresenting for IVF for carriage of the M2/ANXA5 haplotype ought to beconsidered as routine and early in the diagnostic work up of the couplebeing treated with their own gametes. The M2 haplotype appears to be anadditional independent factor that contributes to the risk of pregnancyfailure.

Example 8: Initial Outcome Data for M2 Haplotype Patients Treated withLMWH

Patients were tested for the M2 haplotype and placed into two groups.One group, termed “treated,” consisted of 63 patients. These patientswere both tested for the M2 haplotype carrier status and were treatedwith LMWH at the time of embryo transfer. The second group, termed“untreated,” comprised 62 patients and they were tested for the M2haplotype but not treated with LMWH. In comparing the treated anduntreated groups, the untreated group was one full year younger (35)than the treated (36.1), and the untreated also had infertility for onefull year less (4.24) than the treated group (5.24). See Table 5. Inaddition, 15 patients in the treated group had time lapse embryoculture, known as embryoscope, while only 1 patient in the untreatedgroup had an embryoscope. The treated group also had 7 of the 15patients with embryo time lapse culture progress to clinical pregnancyand live birth. However, both groups shared two characteristics. First,the untreated group had an average of 0.38 previous miscarriages, andthe treated group had an average of 0.52 previous miscarriages. Second,the untreated group had an average of 2.7 previous IVF cycles, and thetreated group had an average of 2.6 previous IVF cycles. Even with theaforementioned minimal differences and close similarities, the treatedgroup had an elevated live birth rate of 38% compared to the globalaverage of 30-35%, while none of the patients (0%) of the untreatedgroup achieved a live birth.

TABLE 5 Preliminary Outcome Data ANXA5 M2 Treated Patients M2 UntreatedPatients M2 positive M2 positive and treated but who had an IVFtreatment before with heparin tested and therefore NO heparin SampleSize (n) 63 42 Mean # Previous IVF Treatments 2.60 2.7 Last TreatmentClinical Pregnancy 28 (44.4%) Event Count Ongoing pregnancy (>24 weeks)9 Deliveries 15 (23.8%) Ongoing (>22 wks)/LB 24 (38%    CP/LBR) LastTreatment Miscarriages Count 4 (14%) Mean age (at date of treatment)36.13 35.0 Average previous miscarriages 0.52 0.38 Average yearsinfertile 5.24 4.24 No of patients with embryos 63 38 90.5% transferredTotal embryos transferred 104 59 Mean age 34.9 (Mean age NOT pregnant34.9) Mean embryos per patient 1.65 1.4 Total with embryoscope 15 23.8%1  2.6% Total with blastocyst transfer 15 23.8% 9 23.7% Positivepregnancy test 35 35/63 = 55.6% 15 15/38 = 39.5% Biochemical pregnanciesas 7 Incidence (7/63 8 Incidence (8/38 positive an endpoint positivepregnancy pregnancy tests 21%) tests (11%) Clinical Pregnancy (CP) 2828/63 = 44.4% 7  7/38 = 18.4% Implantation Rate 55.6% 35/63 = 55.6%39.4% 15/38 = 39.4% Miscarriage 4  4/63 = 6.3% 7  7/38 = 18.4% LiveBirths to date (Aug 2014) 15 15/63 = 38% 0  0/38 = 0%

Thus, it is believed that mothers treated with an anticoagulant(preferably, LMWH) where either the mother or father are carriers forthe M2 haplotype, will have improved clinical pregnancy rates and/orimproved live birth rates. In preferred embodiments, the mother istreated at the time, or very close to, or shortly after, the time ofembryo transfer. LMWH is administered for at least 4 weeks, for at least8 weeks, for at least 12 weeks, or for at least 16 weeks. Treatment withan anticoagulant, such as LMWH, has the ability to increase the chanceof clinical pregnancy, and/or decrease the rate of miscarriage andthereby increase the chance of live birth. Our results are in directcontrast to the results obtained by Rodgers. Moreover treatment with ananticoagulant, such as LMWH, may be used to increase the rates ofsuccessful pregnancies and/or live births in mothers undergoing in vitrofertilization.

It is expected that patients where both partners are carriers for M2haplotypes would benefit the most with the treatment of ananticoagulant, such as LMWH, prior to, simultaneously, and/or withinweeks of becoming pregnant.

TABLE 6 SUBGROUP OF BOTH PARTNERS CARRIERS/HOMOZYGOTE AND TREATED WITHHEPARIN (N) Sample Size 8 Mean previous IVF treatments 2.9 Mean age (atdate of treatment) 34.95 No with one or more 3 Foetal Heart beat (FH)Average Previous miscarriages 0 Average years infertile 6.6 larger timeperiod Number with live birth 3 (37.5%)

Example 9: Statistical Analysis of the Outcome Data for M2 HaplotypePatients Treated with LMWH

Final outcome data was statistically analysed for 125 patients with anembryo transfer who were broken into two study groups; 63 (50.4%)patients in a test/treatment group and 62 (49.6%) in a yardstick (orcontrol) group. The final data extended the results reported above forthe interim analysis. Not only did the data set include information onpregnancy outcomes, but additional variables were recorded summarisingthe patients' demographics and treatment histories. The analysis of thisdata set focuses on three endpoints specified: live births; clinicalmiscarriages; and successful implantations.

The definitions of the three endpoints analysed are as follows:

The live births endpoint is a binary variable indicating whether theembryo transfer resulted in a live birth or not. All patients with anembryo transfer were included in the analysis of the live birthsendpoint.

Clinical miscarriage is a binary variable indicating whether the embryotransfer resulted in a clinical miscarriage or a live birth. Only thosepatients that reached the clinical pregnancy stage (i.e., where anultrasound scan detected a foetal heart rate) were included in theanalysis of clinical miscarriage.

For each patient with an embryo transfer, one, two or three embryos weretransferred into the patient. The implantation incidence rate is thendefined as the number of foetal heart rates detected divided by thenumber of embryos transferred. The implantation incidence rate wasconverted to a successful implantation endpoint prior to analysis. Thesuccessful implantation endpoint is a binary variable indicating whetherthe implantation was a success or failure (i.e., foetal heart ratedetected or not) for each embryo transferred. Each embryo transferredwas included in the analysis of successful implantation

In addition to study group (treatment/control), the followingindependent variables (patient demographics and treatment histories)were also studied to determine whether there is an association betweenthe independent variables and with the three endpoints defined above.

Patient Age

-   -   M2 haplotype results (female and male). M2 haplotype results        were obtained for both the patient (female) and their partner        (male). Following the grouping convention applied in Fishel, et        al. (2014) these were categorised as follows:        -   Both carriers;        -   Male carrier only;        -   Female carrier only;        -   One homozygote, one carrier; and        -   One homozygote only.    -   It was found that there were few patients in the ‘One        homozygote, one carrier’ (8 observations) and ‘One homozygote        only’ (4 observations) categories and therefore regrouped these        into a single homozygote category (‘One homozygote only or one        homozygote/one carrier’).    -   Number of embryos transferred. This variable was grouped into        two categories: one for those embryo transfers with 1 embryo        transferred; and one for those embryo transfers with 2 or 3        embryos transferred.

Type of Incubator

Duration of Infertility (Years)

-   -   Number of previous IVF cycles: This variable was grouped into        five categories depending upon whether the patient had 0, 1, 2,        3, or 4 or more previous IVF cycles.    -   Number of previous miscarriages: This variable was grouped into        three categories depending upon whether the patient had 0, 1, or        2 or more previous miscarriages.    -   Embryo transfer: For each embryo transferred, the type of embryo        was recorded. These were recorded as: morula, blastocyst, or the        number of cells transferred. Given the large number of        categories observed, a single variable was created, grouping the        embryo transfers into those that were blastocyst transfers and        those that were not. Since blastocyst transfers were consistent        across embryos within a transfer, only a single variable is        needed (regardless of the number of embryos transferred).    -   Use of intralipids: Thirty-three embryo transfers in the        treatment group were associated with patients receiving        intralipids. It was determined that for the majority of cases        these patients were given intralipids because they had        immunological problems and that this was more likely to be        associated with pregnancy outcomes. Therefore, these patients        were grouped into those who were treated with intralipids for        immunological problems and those who were not. Two patients were        identified as receiving intralipids for no clinical reason (two        patients) and were therefore not included in the ‘Treated with        intralipids for immunological problems’ category.

Donor Egg Use

For each endpoint, individual tests of association (via univariateregression models) were carried out with the independent variables inorder to assess their statistical significance as individual predictorsof the outcome. All three of the endpoints analysed are binaryvariables. In this case, a logistic regression model is appropriate,which uses a logistic transformation to express the probability of theoutcome (e.g., live birth) as a linear function of the independentvariables.

Following the univariate tests, a multiple logistic regression model isapplied to assess the collective predictive accuracy of the independentvariables for the outcome. This allows for the investigation of thepotential effect of study group on the chance of having each outcome andalso account for (and estimate the effects of) the other independentvariables.

Only those variables that are found to be statistically significant inthe univariate analysis (for at least one odds ratio with a 10%significance level, i.e. requiring the probability that the observedeffect is due to chance alone is less than 10%) are considered forinclusion in this multiple variable model.

For the multiple logistic regression modelling, a backward stepwiseregression algorithm, which begins with the model including allindependent variables that were identified as significant from theunivariate tests, and then successively removes them from the model inorder to determine the model that provides the best fit was used. Themodel fit is determined using likelihood ratio tests (with a 5%significance level, i.e., requiring the probability that the observedeffect is due to chance alone is less than 5%) ensuring that onlyvariables that have a substantial effect on the performance of the finalmodel are included.

Out of the 125 patients with an embryo transfer recorded, there were 34patients who provided an observation in both study groups. This meansthere were patients who underwent IVF twice in the study, once withoutthe study treatment and once with the treatment. The multiple pregnancyoutcomes for these patients are not independent and therefore cannot betreated as such within the statistical model. To account for therepeated nature of the data (multiple observations per patient),logistic mixed-effects models are applied, including a random effect foreach patient in order to model the correlation among their multipleresponses.

Separation was also identified in a number of instances. Separationoccurs in logistic regression when the binary outcome can be separatedby an independent variable. Complete separation occurs when theseparation is perfect whereas quasi-complete separation happens when theoutcome is separated to a certain degree, for example where all of theresponses for one factor of a categorical variable (rather than allfactors) have the same outcome (Heinze & Schemper, 2002). As describedbelow, in this study live births only occurred in patients within thetreatment group and not at all in the control group. Thus, the binaryoutcome‘live birth’ is separated by the independent variable, ‘studygroup’. Similarly, all patients in the control group that reached theclinical pregnancy stage had clinical miscarriages and so ‘clinicalmiscarriage’ is also separated by the independent variable, ‘studygroup’.

In the presence of separation, standard logistic regression modelsfitted via maximum likelihood can produce infinite or biased estimates.Separation is a common problem in logistic regression and is more likelyto occur with smaller sample sizes, with more dichotomous covariates,and with more extreme odds ratios and with larger imbalances in theirdistribution.

There are a few options for dealing with this in the analysis. Firstly,those cases causing separation could be omitted from the analysis.However, this would not be appropriate in this case as it would meanthat information about the effect of this important independent variablewould not exist and also it would not allow for the adjustment of theeffects of the other independent variables for the effect of thisvariable. Furthermore, this would mean throwing away data, reducing thepredictive power of the modelling.

Therefore, two other alternatives are either application of Firth's biasreducing, penalised maximum likelihood logistic regression (Fisher,1992, 1993) or a Bayesian logistic mixed-effect model (Fong, et al.,2010; Zhao, et al., 2006). In this instance the latter was chosen, forthe Bayesian approach provided a more flexible framework that to dealwith the separation issue but also to include a random effect in themodel to account for the repeated observations from some patients, asdiscussed above. It's not possible to incorporate random effects withinFirth's logistic regression model. Using a Bayesian logisticmixed-effect model does require specifying prior distributions for thefixed and random effects. In this case, with no other informationavailable, a Normal distribution was chosen for the fixed effects andthe default flat prior for the random effects.

The successful implantation endpoint did not exhibit separation with anyof the independent variables considered. However, for consistency withthe other outcomes, Bayesian logistic mixed-effect modelling was alsoused for this endpoint. A more standard approach in this case would havebeen a standard logistic mixed-effect analysis. The analysis was re-runusing this approach and found that it produced consistent results withthe Bayesian model.

All analyses were performed in the statistical software package Rversion 3.1.1 (R Core Team, 2013). The bglmer function in the blmepackage was used to implement the Bayesian logistic mixed-effectmodelling (Dorie, 2014).

Live Births

As shown in Table 7, the pregnancy outcomes of the 125 patients with anembryo transfer recorded in the study split by study group. Of the 125patients with an embryo transfer recorded, surprisingly 25/63 (39.7%)within the treatment group resulted in a live birth and none (out of 62)in the control group. The data shows that the odds of a successful livebirth are estimated to be approximately 56 times higher (OR=56.08; 95%CI: 4.95, 635.64) for patients in the treatment group compared with thecontrol group (p=0.0012).

TABLE 7 Pregnancy outcomes for patients with an embryo transfer, bystudy group. Study Group Treatment Control Patients with an embryotransfer 63 62 Number of embryos transferred 104 95 Patients withbiochemical pregnancies 35 23 (positive pregnancy tests) Patientswithout biochemical pregnancies 28 39 (negative pregnancy tests; losspre-implantation) Number of foetal heart rates detected 36 12 Patientswith clinical pregnancies 28 12 (foetal heart rate detected) Patientswithout clinical pregnancies 35 50 Patients with a biochemical loss 7 11(biochemical pregnancies but no foetal heart rate detected) Patientswith clinical miscarriages 3 12 (miscarriages after foetal heart ratedetected) Patients with live births 25 0 Patients with no live births 3862

Moreover, the odds of a successful live birth are estimated to be 2.7times higher (OR=2.69; 95% CI: 1.00, 7.23) for embryo transfers wheretwo or more embryos were transferred compared with embryo transferswhere one embryo was transferred (p=0.050).

TABLE 8 Cross-tabulation of live births versus number of embryostransferred, n (column %). No. Embryos Transferred 1 2/3 Patients withlive births  6 (11.3) 19 (26.4) Patients with no live births 47 (88.7)53 (73.6) Total 53 (100) 72 (100)

Additionally, the number of previous IVF cycles was positivelycorrelated with the odds of a successful live birth. The odds wereestimated to be 4 times higher (OR=4.04; 95% CI: 0.89, 18.37) forpatients with two previous rounds compared to none (p=0.0711); and 7.5times higher (OR=7.46, 95% confidence interval: 1.66, 33.55) forpatients with three previous rounds compared to none (p=0.0088).

TABLE 9 Cross-tabulation of live births versus previous IVF cycles, n(column %). Previous IVF Cycles 0 1 2 3 4+ Patients with 2 (5.9)  6(19.4)  7 (26.9)  9 (40.9) 1 (8.3) live births Patients with 32 (94.1)25 (80.6) 19 (73.1) 13 (59.1) 11 (91.7) no live births Total 34 (100) 31(100) 26 (100) 22 (100) 12 (100)

Furthermore, the odds of a successful live birth are also estimated tobe 2.4 times higher (OR=2.37, 95% CI: 0.91, 6.15) for patients with oneprevious miscarriage compared to none (p=0.0773).

TABLE 10 Cross-tabulation of live births versus previous miscarriages, n(column %). Previous Miscarriages 0 1 2+ Patients with live births 12(14.3) 10 (29.4) 3 (42.9) Patients with no live births 72 (85.7) 24(70.6) 4 (57.1) Total 84 (100) 34 (100) 7 (100)

The use of intralipids (due to immunological problems) was associatedwith 3 times the odds (OR=3.02, 95% CI: 1.19, 7.64) of a successful livebirth compared to a patient not on intralipids (p=0.0199). As shown inTable 11, the proportion of patients with a live birth for those usingintralipids (due to immunological problems) was 35.5%, compared with14.9% for those not using intralipids. However, all of those patients onintralipids were in the treatment group. Looking at only those patientsin the treatment group, also shown in Table 11, these proportionsbecome: 35.5% versus 43.8%, respectively.

TABLE 11 Intralipid Use (due to immunological problems) Yes (%) No (%)Cross-tabulation of Patients with live births 11 (35.5) 14 (14.9) livebirths versus intralipid use due Patients with no 20 (64.5) 80 (85.1) tolive births immunological Total 31 (100) 94 (100) problems, n (column%). Cross-tabulation of Patients with live births 11 (35.5) 14 (43.8)live births versus intraplid use due Patients with no 20 (64.5) 18(56.3) to live births immunological Total 31 (100) 32 (100) problems, n(column %), for patients in the treatment group only.

Donor egg use was associated with 4 times the odds (OR=4.03, 95% CI:0.93, 17.46) of a successful live birth compared to a patient not usinga donor egg (p=0.0622).

TABLE 12 Cross-tabulation of live births versus Donor egg use, n (column%). Donor Egg Use Yes No Patients with live births 4 (50.0) 21 (17.9)Patients with no live births 4 (50.0) 96 (82.1) Total 8 (100) 117 (100) 

Donor egg use was associated with 4 times the odds (OR=4.03, 95% CI:0.93, 17.46) of a successful live birth compared to a patient not usinga donor egg (p=0.0622).

Hence, the data suggest that the study group (e.g., treatment at embryotransfer/implantation with low molecular weight heparin) is astatistically significant predictor for live births, with the odds of alive birth estimated to be approximately 56 times higher for patients inthe test/treatment group compared to the control group (OR=56.08; 95%CI: 4.95, 635.64; p=0.0012). If the odds of a live birth in the controlgroup were 1 to 89 (i.e., p=1/90=1.11% and 1-p=89/90=98.89%; based onthe model estimated odds), with OR=56, the odds of a live birth for thetreatment group would be 56 times as good or approximately 1 to 1.59(i.e., p=34.7/90=38.6% and 1-p=55.3/90=61.4%). So, on average, for everypregnancy in the control group that results in a successful pregnancy,89 will not, but for every pregnancy in the treatment group that resultsin a successfully pregnancy, only 1.59 on average will not.

Clinical Miscarriages

Of the 40 patients with an embryo transfer that reached the clinicalpregnancy stage (i.e., foetal heart rate detected on an ultrasoundscan), 12/12 (100%) of those in the control group resulted in a clinicalmiscarriage and 3/28 (10.7%) of those in the treatment group. The oddsof a clinical miscarriage are estimated to be 0.01 times (i.e., 99%)lower (OR=0.010; 95% CI: 0.001, 0.135) for patients in the treatmentgroup compared with the control group (p=0.0005).

Table 13 Cross-tabulation of clinical miscarriages versus study group, n(column %). Study Group Treatment Control Patients with live births 25(89.3) 0 (0) Patients with clinical miscarriages 3 (10.7) 12 (100) Total28 (100)  12 (100)

Hence, the data suggest that study group (e.g., treatment with lowmolecular weight heparin) is a statistically significant predictor forclinical miscarriage (given clinical pregnancy, i.e., foetal heart ratedetected), with the odds of a clinical miscarriage estimated to be 0.01times (i.e., 99%) lower for patients in the test/treatment groupcompared to the yardstick group (OR=0.010; 95% CI: 0.00079, 0.135;p=0.0005).

Successful Implantation

Of the 199 embryos transferred, 36/104 (34.6%) of those in the treatmentgroup resulted in a successful implantation (foetal heart rate detected)and 12/95 (12.6%) of those in the control group. Thus, the odds of asuccessful implantation are estimated to be 4.1 times greater (OR=4.08;95% CI: 1.85, 8.97) for embryo transfers in the treatment group comparedwith the control group (p=0.0005).

TABLE 14 Cross-tabulation of successful implantation versus study group,n (column %). Study Group Treatment Control Embryos transferred withfoetal 36 (34.6) 12 (12.6) heart rate detected Embryos transferred withfoetal 68 (65.4) 83 (87.4) heart rate not detected Total 104 (100)  95(100)

Hence, the data suggest that study group is a statistically significantpredictor for successful implantation (i.e., foetal heart rate detected,given embryo implanted), with the odds of successful implantationestimated to be approximately 4 times higher for patients in thetest/treatment group compared to the control group (OR=4.08; 95% CI:1.85, 8.97; p=0.0005).

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What is claimed:
 1. A method of treating a M2 haplotype pregnancy,comprising: a. identifying a M2 haplotype pregnancy, wherein said M2haplotype pregnancy exists when either the biological mother or thebiological father is a carrier of the ANXA5 M2 haplotype; b.administering to the mother of a M2 haplotype pregnancy an effectiveamount of an anticoagulant.
 2. The method of claim 1, wherein both thebiological mother and the biological father is a carrier of the ANXA5 M2haplotype.
 3. The method of either claim 1 or claim 2, wherein theanticoagulant is administered after the mother is pregnant.
 4. Themethod of either claim 1 or claim 2, wherein the anticoagulant isadministered at the same time or closely after the time of embryotransfer.
 5. The method of either claim 1 or claim 2, wherein theanticoagulant is administered prior to pregnancy.
 6. The method of anyone of claims 1-5, wherein the anticoagulant is LMWH.
 7. The method ofany one of claims 1-6, wherein the mother is undergoing in vitrofertilization or intrauterine insemination.
 8. A method of reducingobstetrics complications comprising: a. identifying a M2 haplotypepregnancy, wherein said M2 haplotype pregnancy exists when either thebiological mother or the biological father is a carrier of the ANXA 5 M2haplotype; b. administering to the mother of a M2 haplotype pregnancy aneffective amount of an anticoagulant, wherein said anticoagulant reducesthe risk of obstetrics complications.
 9. The method of claim 8, whereinboth the biological mother and the biological father is a carrier of theANXA5 M2 haplotype.
 10. The method of either claim 8 or claim 9, whereinthe anticoagulant is administered soon after the mother is pregnant. 11.The method of either claim 8 or claim 9, wherein the anticoagulant isadministered at the same time of embryo transfer or intrauterineinsemination.
 12. The method of either claim 8 or claim 9, wherein theanticoagulant is administered prior to pregnancy.
 13. The method of anyone of claims 8-12, wherein the anticoagulant is LMWH.
 14. The method ofany one of claims 8-13, wherein the mother is undergoing in vitrofertilization or intrauterine insemination.
 15. The method of any one ofclaims 8-14, wherein the obstetrics complications are recurrentpregnancy loss (RPL), infertility, miscarriage, in vitro fertilization(IVF) failure, IUI failure, implantation failure, foetal growthrestriction (FGR), small for gestational age (SGA) newborn,intra-uterine foetal death (IUFD), gestational hypertension (GH),pre-eclampsia (PE) and/or venous thromboembolism (VTE).
 16. The methodof claim 15, wherein the infertility is selected from unexplained maleinfertility, unexplained female infertility, or infertility of unknownorigin
 17. A method of determining a M2 haplotype pregnancy, comprising:a. identifying the M2 haplotype carrier status either the biologicalmother or the biological father, wherein said identification isdetermined based on genomic analysis; b. recording the M2 haplotypecarrier status of the biological mother and the biological father; c.reporting whether a M2 haplotype pregnancy exists if either thebiological mother or the biological father is a carrier of the ANXA5 M2haplotype.
 18. The method of claim 17, wherein both the biologicalmother and the biological father is a carrier of the ANXA5 M2 haplotype.19. The method of either claim 17 or 18, wherein the mother isundergoing in vitro fertilization or IUI.
 20. The method of any one ofclaims 17-19, wherein said identification uses PCR or sequencingtechniques.
 21. The method of any one of claims 17-20, wherein saididentification uses IHG technology
 22. The method of any one of claims17-21, wherein a fluorescent molecule is used to detect the ANXA5 M2haplotype.
 23. The method of any one of claims 17-22, wherein the methodof detection uses a solid phase.
 24. The method of claim 23, wherein thesolid phase is a bead or a plate.
 25. The method of either claim 23 or24, wherein the solid phase comprises plastic, silicon, glass,polystyrene, aluminium, steel, iron, copper, nickel, silver, gold,nitrocellulose or nylon.
 26. The method of any one of claims 17-25,wherein an oligomer having a length at least 15 nucleotides is used todetect the ANXA5 M2 haplotype.
 27. The method of any one of claims17-26, wherein said method utilizes Next Generation Sequencingtechniques.
 28. A method of increasing the rate of pregnancy comprising:a. identifying a M2 haplotype pregnancy, wherein said M2 haplotypepregnancy exists when either the biological mother or the biologicalfather is a carrier of the ANXA 5 M2 haplotype; b. administering to themother of a M2 haplotype pregnancy an effective amount of ananticoagulant, wherein said anticoagulant increases the rate ofpregnancy.
 29. The method of claim 28, wherein both the biologicalmother and the biological father is a carrier of the ANXA5 M2 haplotype.30. The method of either claim 28 or claim 29, wherein the anticoagulantis administered soon after the mother is pregnant.
 31. The method ofeither claim 28 or claim 29, wherein the anticoagulant is administeredat the same time of embryo transfer or IUI.
 32. The method of eitherclaim 28 or claim 29, wherein the anticoagulant is administered prior topregnancy.
 33. The method of any one of claims 28-32, wherein theanticoagulant is LMWH.
 34. The method of any one of claims 28-32,wherein the mother is undergoing in vitro fertilization or IUI.
 35. Themethod of claim 34, wherein the mother has an increased risk of anobstetrics complication selected from recurrent pregnancy loss (RPL),infertility, miscarriage, in vitro fertilization (IVF) failure, IUIfailure, implantation failure, foetal growth restriction (FGR), smallfor gestational age (SGA) newborn, intra-uterine foetal death (IUFD),gestational hypertension (GH), pre-eclampsia (PE) and/or venousthromboembolism (VTE).
 36. The method of claim 35, wherein theinfertility is selected from unexplained male infertility, unexplainedfemale infertility, or infertility of unknown origin.
 37. The method ofany one of claims 1-16 or 28-36, wherein a therapeutically effectiveamount of an anticoagulant is administered for at least 4 weeks.
 38. Themethod of claim 37, wherein the anticoagulant is administered for atleast 8 weeks.
 39. The method of claim 38, wherein the anticoagulant isadministered for at least 12 weeks.
 40. The method of claim 39, whereinthe anticoagulant is administered for at least 16 weeks.
 41. The methodof claim 40, wherein the anticoagulant is administered until the motherdelivers the baby.
 42. The method of any one of claims 1-16 or 28-41,wherein a therapeutically effective amount of an anticoagulant iseffective to increase the rate of implantation.
 43. The method of anyone of claims 1-16 or 28-41, wherein a therapeutically effective amountof an anticoagulant is effective to decrease the rate of miscarriagebefore detection of a foetal heartbeat.